Topical minocycline compositions and methods of using the same

ABSTRACT

Topical minocycline compositions with reduced fluorescence are provided. In some instances, the compositions include an amount of a minocycline active agent associated with porous calcium particles. Also provided are methods of using the compositions, e.g., in the treatment of acne.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/434,368filed Jan. 19, 2011; the disclosure of which application is hereinincorporated by reference.

INTRODUCTION

Oral tetracycline-class antibiotics are frequently used in the treatmentof acne. Tetracycline-class antibiotics are known to have some sideeffects. These side effects include vestibular symptoms such as vertigo,dizziness or blurred vision. These effects are sometimes disabling. See,Gould & Brookler, Arch. Otolarang. Vol. 96, p. 291 (1972); Williams etal., Lancet, Sep. 28, 1974, p. 144-45; Fanning & Gump, Arch. Intern.Med., Vol. 136, pp. 761-62 (1976). Headache and general malaise, alongwith gastro-intestinal symptoms such as the diarrhea, nausea, gas, orcramps may also occur. Dry nose and dry mouth are also occasionallyencountered. One of the oral tetracycline-class antibiotics used in thetreatment of acne is minocycline hydrochloride. Oral dosage forms ofminocycline hydrochloride are available commercially under various tradenames. The Approved Drug Products with Therapeutic EquivalenceEvaluations (“Orange Book”) lists a number of oral dosage forms ofminocycline hydrochloride that are AB-rated to the MINOCIN® brand ofminocycline hydrochloride. These commercial products areimmediate-release oral dosage forms of minocycline hydrochloride thathave been determined by the Food and Drug Administration (FDA) to betherapeutically equivalent to the MINOCIN® brand of minocyclinehydrochloride on the basis of adequate in vivo and/or in vitro evidencesupporting bioequivalence.

SUMMARY

Topical minocycline compositions are provided. In some instances, thecompositions include an amount of minocycline associated with porouscalcium particles, e.g., via entrapment in the pores of the particlesand/or ionic binding and/or non-covalent binding to the surface of theparticles and/or loosely associated with the particles. Also providedare methods of using the compositions, e.g., in the treatment of acne.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the results of a stability assay reported in theExperimental section below, where the results demonstrate thatminocycline associated with porous calcium phosphate particles exhibitsgreater stability than free minocycline.

FIG. 2 provides the results of a pH release assay reported in theExperimental section below, where the results demonstrate thatminocycline associated with porous calcium particles is released atstratum corneum skin pH conditions and is not degraded when released.

FIG. 3 provides a picture of both free minocycline powder under whitelight and black light. The picture shows that minocycline associatedwith porous calcium phosphate particles exhibits less fluorescence thanfree minocycline.

FIG. 4 provides a picture of silicone oil, silicone oil containing freeminocycline, and silicone oil containing minocycline associated withporous calcium phosphate particles. The picture shows that minocyclineassociated with porous calcium phosphate particles exhibits lessfluorescence than free minocycline.

FIG. 5 graphically provides the results of a dermal delivery assayreported in the Experimental section, below. The results show thatminocycline associated with porous calcium phosphate particles isdelivered in a sustained release manner into the skin.

DETAILED DESCRIPTION

Topical minocycline compositions are provided. In some instances, thecompositions include an amount of minocycline associated with porouscalcium particles, e.g., via entrapment in the pores of the particlesand/or ionic binding and/or non-covalent binding to the surface of theparticles and/or loosely associated with the particles. Also providedare methods of using the compositions, e.g., in the treatment of acne.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Methods recited herein may be carried out in any order of the recitedevents which is logically possible, as well as the recited order ofevents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Topical Minocycline Compositions

As summarized above, topical minocycline compositions are provided. Thecompositions of the invention are formulated for delivery of aminocycline active agent to a topical location of a subject, such as akeratinized skin surface of a mammalian subject, such as a humansubject. By keratinized skin surface is meant a skin location of asubject, i.e., a location of the external covering or integument of ananimal body. Because the compositions of the invention are formulatedfor delivery to topical locations, they are formulated so as to bephysiologically compatible with the topical location for which they areformulated. Accordingly, when contacted with the target keratinized skinsurface for which they are formulated, the formulations do not causesubstantial, if any, physiological responses (such as inflammation orirritation) that would render the use of the formulations unsuitable fortopical application.

In some instances, the topical minocycline compositions are reducedfluorescence compositions. As such, these compositions exhibit reduced,if any, minocycline-generated fluorescence upon excitation with light ofan appropriate wavelength, e.g., 365 nm, following topical application.By reduced fluorescence is meant 5-fold or less fluorescence, such as10-fold or less fluorescence, including 15-fold or less fluorescence,20-fold or less fluorescence, 25-fold or less fluorescence, 30-fold orless fluorescence, as compared to a control formulation, e.g., water andminocycline hydrochloride. In some instances, the compositions exhibitno fluorescence (e.g., fluorescence is undetectable with naked eye).

As summarized above, compositions of the invention include an amount ofa minocycline active agent associated with porous calcium containingparticles. As the minocycline active agent is associated with porouscalcium particles, it may be entrapped in the pores of the particlesand/or ionically bound to the particles and/or non-covalently bound tothe particles and/or loosely associated with the particles. As such, theparticles may be described as being loaded with an amount of minocyclineactive agent. By “loaded” is meant that the particles include an amountof minocycline active agent that is associated with the particles. Asthe minocycline active agent is associated with the particles, theminocycline active agent does not dissociate from the particles in anysubstantial amount when the particles are present in the deliverycomposition prior to use. Because substantially none of the minocyclineactive agent dissociates from the particles, any amount that doesdissociate is 40% or less, for example 20% or less, such as 10% or less,including 5% or less of the originally associated amount of minocyclineactive agent. The amount of minocycline active agent component that isassociated with the calcium particles may vary, and in certainembodiments ranges from 0.01 mg/g to 1000 mg/g, such as from 0.1 mg/g to700 mg/g and including 1 mg/g to 300 mg/g minocycline active agent/gramparticles.

As described above, the topical minocycline compositions of interestinclude an amount of minocycline active agent associated with porouscalcium particles. In some instances, the topical compositions includean amount of a UV quencher and/or emission extinguisher and/or both. Thetopical compositions may further include various delivery vehicles, aswell as other components, as desired. These aspects, and others, of thecompositions are now reviewed in greater detail.

Porous Calcium Particles

Particles employed in methods of the invention are porous calciumphosphate particles. By “porous” is meant that the particles have aporosity of 30% or more, such as 40% or more, including 50% or more,where the porosity may range from 30% to 200% or more, such as from 40%to 150%, including from 45% to 100%, as determined using a mercuryintrusion porosimeter porosity determination protocol as described inASTM D 4284-88 “Standard Test Method for Determining Pore VolumeDistribution of Catalysts by Mercury Intrusion Porosimetry”. Porosity isalso described by “pore volume (ml/g)” and in such instances many rangefrom 0.01 ml/g to 4.0 ml/g. In some cases, the particles have a porositysuch that their internal surface area ranges from 10 m²/g to 150 m²/g,such as from 20 m²/g to 100 m²/g, including 30 m²/g to 80 m²/g, asdetermined using a BET gas adsorption surface area determinationprotocol as described in ASTM D3663-03 Standard Test Method for SurfaceArea of Catalysts and Catalyst Carriers. The pore diameter may vary,ranging in certain instances from 2 to 500 nm, such as 5 to 200 nm,including 10 to 100 nm. In addition, the particles may have a tappingdensity ranging from 0.2 g/cm³ to 0.5 g/cm³, such as from 0.25 g/cm³ to0.45 g/cm³, including from 0.3 g/cm³ to 0.4 g/cm³. The tap density canbe measured by using standard ASTM WK13023—New Determination of TapDensity of Metallic Powders by a Constant Volume Measuring Method.

In some instances, the particles are rigid particles which are uniformand spherical in shape. By “rigid” is meant that the particles are hard,such that they are not pliant. By “uniform” is meant that the shape ofthe particles does not vary substantially, such that the particles havesubstantially the same spherical shape. The term “spherical” is employedin its conventional sense to mean a round body whose surface is at allpoints substantially equidistant from the center. Of interest in certainembodiments are calcium phosphate particles in which the median diameteris 20 μm or less, such as 10 μm or less, including 5 μm or less, wherein some instances the medium diameter is 4 μm or less, such as 3 μm orless, including 2 μm or less.

The particles are, in some instances, chemically pure. By chemicallypure is meant that the particles are made up of substantially one typeof compound, e.g., a calcium compound, such as a calcium phosphatemineral. Of interest as porous particles are calcium containingparticles, such as calcium containing particles that are made of amolecule that includes calcium cation and a suitable anion, e.g.,carbonate, phosphate, etc. In some instances, the particles are calciumcarbonate particles, such as but not limited to the calcium carbonateparticles disclosed in U.S. Pat. Nos. 5,292,495 and 7,754,176. In someinstances, the calcium phosphate particles are made up of a calciumphosphate that is described by the molecular formula Ca₁₀(PO₄)₆(OH)₂.

In some instances, the particles are ceramic particles. By ceramic ismeant that the particles are produced using a method which includes astep of subjecting the particles to high temperature conditions, wheresuch conditions are illustrated below. High temperatures may range from200 to 1000° C., such as 300 to 900° C. and including 300 to 800° C. Insome embodiments, the particles have a compression rupture strengthranging from 20 to 200 MPa, such as from 50 to 150 MPa, and including 75to 90 MPa, as determined using a SHIMADZU MCT-W500 micro-compressiontesting machine particle strength determination protocol with a particlesintered at temperature of 400° C. to 900° C., as described in EuropeanPatent EP1840661. In some embodiments, the particles are biodegradable,by which is meant that the particles degrade in some manner, e.g.,dissolve, over time under physiological conditions. As the particles ofthese embodiments are biodegradable under physiological conditions, theyat least begin to dissolve at a detectable rate under conditions of pHof 5.8 or less, such as 5.5 or less, e.g., 5.3 or less, including 5 orless, e.g., 4.9 or less.

The porous calcium phosphate particles employed in embodiments of themethods may be prepared using any convenient protocol. In one protocolof interest, the particles are manufactured by spray drying a slurrywhich includes porous calcium phosphate (e.g., hydroxyapatite) crystals(which may range from 2 nm to 100 nm size range) to produce porouscalcium phosphate particles. The resultant particles are then sinteredfor a period of time sufficient to provide mechanically and chemicallystable rigid spheres. In this step, the sintering temperatures may rangefrom 100° C. to 1000° C. for a period of time ranging from 1 hour to 24hours or more.

Minocycline Active Agent

As summarized above, the compositions of these embodiments include aminocycline active agent. The minocycline active agent may be in theform of a free base, an acid salt (e.g., hydrochloride salt) or amixture thereof. Reference herein to “minocycline” will be understood asencompassing all such forms, unless the context dearly indicatesotherwise. An example of a minocycline salt of interest is minocyclinehydrochloride (HCl), which has the structure:

Dosages of minocycline salts will be understood to be on the basis ofthe amount of minocycline free base provided thereby, and thus may beexpressed as a minocycline free base equivalent dosage or amount.Minocycline salts are pharmaceutically acceptable in some embodiments.The term “pharmaceutically acceptable”, as used herein, refers to adrug, salt, carrier, etc., that can be introduced safely into an animalbody (e.g., taken orally and digested, etc.).

UV Quenchers/Emission Extinguishers

The topical compositions may include an amount of a UV quencher/emissionextinguisher. UV quenchers/emission extinguishers of interest includebut are not limited to: Allantoin PABA, Benzalphthalide, Benzophenone-1,Benzophenone-2, Benzophenone-3, Benzophenone-4, Benzophenone-5,Benzophenone-6, Benzophenone-7, Benzophenone-8, Benzophenone-9,Benzophenone-10, FD&C Blue No. 1, FD&C Blue No. 2, D&C Blue No. 4, D&CBlue No. 9, FD&C Green No. 3, D&C Green No. 5, D&C Green No. 6, D&CGreen No. 8, D&C Orange No. 4, D&C Orange No. 5, D&C Orange No. 10,Benzophenone-11, Benzophenone-12, Benzyl Salicylate, Benzylidene CamphorSulfonic Acid, Bornelone, Bumetrizole, Butyl methoxydibenzoylmethane,Butyl PABA, Cinoxate, DEA-Methoxycinnamate, Dibenzoxazoyl Naphthalene,Diisopropyl Ethyl Cinnamate, Diisopropyl Methyl Cinnamate,Dimorpholinopyridazinone, Drometrizole, Esculin, Ethyl DihydroxypropylPABA, Ethyl Diisopropylcinnamate, Ethylhexyl Dimethyl PABA, D&C OrangeNo. 11, FD&C Red No. 3, FD&C Red No. 4, D&C Red No. 6, D&C Red No. 7,D&C Red No. 17, D&C Red No. 21, D&C Red No. 22, D&C Red No. 27, D&C RedNo. 28, D&C Red No. 30, D&C Red No. 31, Ethylhexyl Methoxycinnamate,Ethylhexyl Salicylate, Ethylhexyl Triazone, Ethyl Methoxycinnamate,Ethyl PABA, Ethyl Urocanate, Etocrylene, Glyceryl EthylhexanoateDimethoxycinnamate, Glyceryl PABA, Glycol Salicylate, Homosalate,Isoamyl Cinnamate, Isopropylbenzyl Salicylate, IsopropylMethoxycinnamate, Menthyl Anthranilate, Menthyl Salicylate,Methylbenzylidene Camphor, Octocrylene, Octrizole, PABA,Phenylbenzimidazole Sulfonic Acid, Polyacrylamidomethyl BenzylideneCamphor, D&C Red No. 33, D&C Red No. 34, D&C Red No. 36, D&C Red No. 39,D&C Violet No. 2, FD&C Yellow No. 5, FD&C Yellow No. 6, D&C Yellow No.7, Ext. D&C Yellow No. 7, D&C Yellow No. 8, D&C Yellow No. 10, D&CYellow No. 11, Potassium Methoxycinnamate, Potassium PhenylbenzimidazoleSulfonate, Red Petrolatum, Sodium Urocanate,

TEA—Phenylbenzimidazole Sulfonate, TEA-Salicylate, TerephthalylideneDicamphor Sulfonic Acid, Titanium Dioxide, Urocanic Acid, Zinc CeriumOxide.

Coating

Where desired, the minocycline associated calcium particles may includea coating. Coating materials (which may include one or more coatingmaterial) of interest are those that may preserve the association of theminocycline active agent with the calcium phosphate particles in variousformulations, e.g. formulations designed for topical application to theskin. Suitable coating agents include agents that are physiologicallyacceptable and are solid at room temperature and are suitable forapplication to the skin. The coating material component may be a singlematerial or a combination of two or more materials, e.g., where thecombination provides for one or more desirable properties.

Materials that find use as coating materials include, but are notlimited to waxes, butters, etc., where specific coating materials ofinterest include: Acrocomia Aculeata Seed Butter, Almond Butter, AloeButter, Apricot Kernel Butter, Argan Butter, Attalea Maripa Seed Butter,Avocado Butter, Babassu Butter, Bacuri Butter, Bagura Soft Butter,Baobab Soft Butter, Bassia Butyracea Seed Butter, Bassia Latifolia SeedButter, Black Currant Seed Butter, Brazil Nut Butter, Camelina Butter,Camellia Butter, Candelilla Butter, Carnauba Butter, CarpotrocheBrasiliensis Seed Butter, Chamomile Butter, Cocoa Butter, CoconutButter, Coffee Butter, Cotton Soft Butter, Cranberry Butter, CupuacuButter, Grape Seed Butter, Hazel Nut Butter, Hemp Seed Butter, HorsetailButter, Illipe Butter, Irvingia Gabonensis Kernel Butter, Jojoba Butter,Karite Butter, Kokum Butter, Kukui Butter, Lavender Butter, LemonButter, Lime Butter, Macadamia Butter, Mango Butter, Marula Butter,Monoi Butter, Mowrah Butter, Mucaja Butter, Murumuru Butter, OleaButter, Olive Butter, Orange Butter, Palm Oil, Passion Butter, PhulwaraButter, Pistachio Butter, Pomegranate Butter, Pumpkin Butter, RaspberryButter, Rice Butter, Sal Butter, Sapucainha Butter, Sesame Butter, SheaButter, Soy Butter, Tamanu Butter, Sunflower Seed Butter, Sweet almondButter, Tangerine Butter, Tucuma Seed Butter, Ucuuba Butter, Wheat GermButter, acrawax, bayberry wax, beeswax, candelilla wax, castor wax,carnauba wax, ceresin wax, esparto wax, glycowax, jojoba wax, Japan wax,lignite wax, linear polyethylene wax, microcrystalline petroleum wax,montan wax, ouricouri wax, ozokerite wax, paraffin wax, rice bran wax,shellac wax, silicone waxes, synthetic waxes, sugarcane wax, petrolatum,hard tallow, cetyl alcohol, lanolin alcohol, lanolin, stearyl alcohol,stearone, glyceryl monostearate, glyceryl distearate, glycerolpalmitostearate, cetyl palmitate, ethylcellulose, acrylic resins,dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride,polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3butylene glycol, ethylene homopolymers, ethylene-propylene copolymers,ethylene-hexene copolymers, ethylene glycol methacrylate, and/orpolyethylene glycols, such as PEG-18, PEG-20, PEG-32, PEG-75, PEG-90,PEG-100, and PEG-180.

Delivery Component

The topical compositions of the invention may include an amount of theminocycline active associated calcium particles combined with a deliverycomposition component. The delivery composition component refers to thatportion of the non-fluorescent topical composition that is not theminocycline active agent bound particles. The amount of minocyclineactive agent associated particles that is present in the deliverycomposition and therefore combined with a delivery composition componentmay vary. In some embodiments, the amount of minocycline active agentassociated with particles that is present in the delivery compositionand therefore combined with other delivery composition components mayvary. In some embodiments the amount of minocycline active agentassociated with particles ranges from 0.01 to 200 mg/g, such as 0.1 to100 mg/g and including 1 to 50 mg/g active agent bound particles pergram of delivery composition component.

Delivery vehicles of interest include, but are not limited to,compositions that are suitable for applications via one or more of oral,topical, implantation, ocular, aural, rectal, vaginal, etc., routes. Incertain embodiments, the vehicle is formulated for application to atopical region or surface of a subject, such as a keratinized skinsurface. The subject compositions may be formulated as stable solutionsor suspensions of the components, e.g., in an aqueous solvent. Wheredesired, the components may be combined with one or more carriermaterials to form a solution, suspension, gel, lotion, serums, cream,ointment, aerosol spray, roll-on, foam products, mousses, powders,sticks, or the like, as desired. Of interest in certain embodiments areaqueous delivery vehicles, i.e. aqueous vehicles that include a certainamount of water.

The topical composition may also contain other physiologicallyacceptable excipients or other minor additives, particularly associatedwith organoleptic properties, such as fragrances, dyes, buffers, coolingagents (e.g. menthol), coating materials or the like.

Lotions (as well as other topical formulations) of interest may includeone or more of the following components: Water, Viscosity modifiers,Humectants, Vegetable oils and hydrogenated vegetable oils, Emollients,Silicone, Conditioning Agents, Emulsifiers, Glyceryl Esters of FattyAcids, Silicone, C1-C30 monoesters and polyesters of sugar, ConditioningAgents, Preservatives, etc. Depending on the topical formulation,additional components of interest include: Abrasives, Absorbents,Antimicrobial and antifungal agents, Astringents, Anti-Acne agents,Anti-wrinkle agents, Anti-oxidants, Antimicrobials, Binders, Biologicalactives, Buffering actives, Bulking actives, Chelating agents, Chemicaladditives, External analgesics, Film former agents, Opacifying agents,pH adjusters, Reducing agents, Colorants, Fragrances, Cosmetic SoothingAgents, Tanning actives & accelerators, Skin lightening/whiteningagents, Sunscreens, Surfactants, Skin Conditioning Agents, Vitamins,etc.

As indicated above, of interest in certain embodiments are semi-soliddelivery compositions, such as gels, creams and ointments. Suchcompositions may be mixtures of (in addition to the active agent) water,water soluble polymers, preservatives, alcohols, polyvalent alcohols,emulsifying agents, wax, solvents, thickeners, plasticizers, pHregulators, water-retaining agents and the like. Furthermore, suchcompositions may also contain other physiologically acceptableexcipients or other minor additives, such as fragrances, dyes, buffers,coating materials or the like.

Methods of Making Delivery Compositions

Aspects of the invention further include methods of making the topicalcompositions. While any convenient fabrication protocol may be employed,in some instances the methods of fabrication include combining an amountof a minocycline active agent associated particles with a deliverycomposition component to produce the desired delivery composition. Theactive agent associated particles may be produced using any convenientprotocol. One protocol of interest includes combining a liquidcomposition of the active agent, such as an aqueous composition of theactive agent, with a liquid composition of the porous calcium particles(with agitation where desired) under conditions sufficient to producethe desired active agent bound particles. Following production of thedesired active agent associated particles, the resultant particles arethen combined with the delivery composition component using anyconvenient protocol. The particular protocol employed may vary dependingon the nature of the delivery composition component, where in certaininstances the delivery composition component and active agent loadedparticles may be combined with mixing to produce the desired deliverycomposition.

Methods of Use

In practicing methods of the invention, a topical minocyclinecomposition is applied to a topical region of a subject and maintainedat the topical region for a period of time sufficient to result in thedesired delivery of the minocycline active agent to the subject, asdescribed above. The topical region is, in certain embodiments, akeratinized skin region. The keratinized skin region, including hairfollicles, sweat glands and sebaceous glands, may be present at avariety of locations, e.g., limbs, arms, legs; torso, e.g., chest, back,stomach; head, e.g., neck, face; etc. In certain embodiments, the regionwill be a head region, such as a facial region, e.g., forehead,occipital region, around the mouth, on the nose, etc. The topical regionto which the composition is applied may vary with respect to area,ranging in certain embodiments from 1 mm² to 300 cm² or more, such asfrom 1 to 50 cm², and including from 3 to 10 cm².

Following application, the composition is maintained at the site ofapplication for a period of time sufficient for a desired therapeuticoutcome to occur, e.g., amelioration of a symptom(s) of interest,reducing acne. The period of time may vary, and in certain embodimentsranges from 1 min to 24 hours or longer, such as from 30 min to 12 hoursand including from 1 hour to 12 hours.

In practicing the subject methods, a subject may be administered asingle dose or two or more doses over a given period of time. Forexample, over a given treatment period of one month, 1 or more doses,such as 2 or more doses, 3 or more doses, 4 or more doses, 5 or moredoses, etc., may be administered to the subject, where the doses may beadministered weekly or daily or even multiple times per day.

Methods of the invention may result in decreased fluorescence of theminocycline active agent when the active agent is associated with porouscalcium particles as compared to a suitable control, such as acomposition made up of the minocycline active agent and water, where theminocycline active agent is not bound to porous calcium particles. Theobserved decrease in fluorescence as compared to a control may have amagnitude of a factor of 2-fold or more, such as 5-fold or more,including 10-fold or more, including 25-fold or more, 50-fold or more or75-fold or more.

Methods of the invention may include a step of diagnosing a subject asbeing a subject in need of topical minocycline administration. Forexample, the methods may include assessing whether a subject issuffering or likely to suffer from acne and determining that topicalminocycline administration is desirable in order to treat the acneand/or prevent occurrence of the acne.

The subject methods and compositions may be used in a variety ofdifferent kinds of animals, where the animals are typically “mammals” or“mammalian,” where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats),lagomorpha (e.g., rabbits) and primates (e.g., humans, chimpanzees, andmonkeys). In certain embodiments, the subjects or patients are humans.

Utility

The compositions and methods of the invention find use in a variety ofdifferent applications in which it is desired to topically deliver aminocycline active agent to a subject. In delivering minocycline activeagents to a topical location of a subject, formulations of the inventionmay deliver the minocycline active agent bound particles at least intoan epidermal location that is beneath the skin surface of a subject. Assuch, embodiments of the invention include methods of deliveringminocycline active agent loaded particles into the stratum corneum of asubject, where the methods may result in delivery of the minocyclineinto the deep stratum corneum and/or dermis of a subject. By “deepstratum corneum” is meant a region that is 2 or more cell layers belowthe skin surface, such as 5 or more cell layers below the skin surface,including 10 or more cell layers below the skin surface. In someinstances, the minocycline is delivered to region of the stratum corneumthat is 2 μm or more, such as 5 μm or more and including 15 μm or morebelow the surface of the skin. In some instances, the minocycline isdelivered to regions below the stratum corneum into the epidermis, suchthat it is delivered to 50 μm or more below the stratum corneum, such as100 μm or more below the stratum corneum and including 180 μm or morebelow the stratum corneum. In some instances, the minocycline isdelivered to regions below the epidermis into the dermis region, suchthat it is delivered 500 μm or more below the epidermis, such as 1000 μmor more below the epidermis and including 1400 μm or more below theepidermis.

Upon contact with the topical location, the minocycline active agentbound particles begin to release their minocycline active agent“payload” and break down (e.g., via dissolution caused by pH gradient ofthe skin), as the porous particles dissociate under acidic conditions,e.g., conditions of pH 6 or lower, such as 5.5 or lower, including 4.90or lower, such as the physiological acidic conditions of the stratumcorneum. The time required for partial dissolution of particles in thestratum corneum may vary, and in certain embodiments ranges from 1minute to 24 hours, such as 10 minutes to 12 hours and including 30minutes to 3 hours, over which time period active agent is released fromthe active agent bound particles. The proportion of active agent that isreleased from the active agent bound particles may vary, and in certaininstances is 1% or more, such as 10% or more, including 50% or more(w/w). When the particles are delivered to skin, the minocycline activeagent component associated with the particle can dissociate from theparticle in amounts ranging from 0.1. to 100%, in some instancesdelivering into the skin 50% or less, 40% or less, 30% or less, 20% orless, 10% or less, 5% or less, including 2% or less of the minocyclineactive agent associated with the particles.

In some embodiments, the compositions and methods are used to treatacne. Acnes of interest include, but are not limited to: acne vulgaris;acne rosacea; acne conglobata; acne fulminans; gram-negativefolliculitis; pyoderma faciale; and combinations thereof. The acne maybe a severe form of acne, a moderate form of acne, or a mild form ofacne, and may include inflammatory and/or non-inflammatory lesions. Insome embodiments, the compositions and methods described herein can beused to treat inflammatory lesions of acne vulgaris.

Effective treatment of acne may be characterized in various ways. Forexample, effective treatment of acne may be characterized as areduction, and in some embodiments a substantial reduction, in thenumber of acne lesions. The acne lesions may be defined as at least oneof inflammatory and non-inflammatory lesions. Effective treatment ofacne may be characterized as a reduction in the severity of acne.Effective treatment of acne may be characterized as a reduction in theduration of an outbreak. For example, a composition described herein mayreduce the duration that a lesion will remain after it has formed.Effective treatment of acne may be characterized as a reducedprobability of an acne-related symptom. For example, a compositiondescribed herein may reduce the probability of developing furtherlesions.

In some embodiments, the acne is at least partially caused by hormonalchanges, excessive production of one or more male hormones, orpregnancy. The acne may be caused by a medication, such as acontraceptive pill, ointments for eczema, or medicine for epilepsy. Theacne may be caused by a drug, such as androgens, lithium, orbarbiturates.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL 1. Materials and Methods

A. Materials

1. Hydroxysomes® (INCI Name: Hydroxyapatite, Laboratory Skin Care, Inc.Lot No. 0712-2701) was used for the binding, releasing and fluorescencestudy.

2. Minocycline Hydrochloride (HCl), 4,7-Bis(dimethylamino)1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-1,1′-dioxo-2-naphthacenecarboxamidemonohydrochloride, Manufacturer Sigma-Aldrich Product No.: M 9511 (LotNo. 078K1053), CAS No.: 13614-98-7

3. 1% w/v minocycline/water, Batch #RNB7132, kept at 4° C. in dark.B. Minocycline HCl Detection by UV-Vis Spectroscopy

The spectra of minocycline solution in water were obtained with Shimadzuspectrophotometer (Model number UV-1650PC, Detection at UV 273 nm,)

C. Binding of Minocycline HCl to Hydroxysomes®

Hydroxysomes® (calcium phosphate particles, Laboratory Skin Care, SanCarlos, Calif.) were suspended in water. Minocycline solution (0.01-20mg/ml in water) was added. The final pH of the suspension was adjustedto 6.2 to 8.0 with acid. The binding suspension was mixed by shaking for30 min.

The binding suspensions were centrifuged at 10,000 rpm for 10 min in thebench top microcentrifuge. The supernatants were analyzed with theShimadzu spectrophotometer to quantify the free minocycline.

The bound minocycline was calculated by subtracting the amount of theminocycline detected in the supernatant, from the total initial amountin the binding suspension. The total initial minocycline absorbance atthe binding pH was calculated from the linearity equation of thestandard plot.

Controls: Minocycline solution at the same pH with the sameconcentration as in the binding mixture.

D. Example Formulation

Nr. Raw Material INCI Name % w/w 1 Purified Water Purified Water 88.67 2Xanthan Gum Xanthan Gum 0.30 3 Glycerin Glycerin 0.99 4 CCTCaprylic/Capric 2.98 Triglyceride 5 Stearyl Alcohol Stearyl Alcohol 1.996 Cetyl Alcohol Cetyl Alcohol 1.99 7 Ritapro 165 Glyceryl Stearate, 0.99PEG-100 Stearate 8 Euxyl PE 9010 Phenoxyethanol, 0.99 Ethylhexylglycerin9 Sepiplus 400 Polyacrylate-13, 0.50 Polyisobutene, Polysorbate 20 10Minocycline HCl- Minocycline HCl, 0.60 Hydroxysomes ™ HydroxyapatiteTOTAL: 100.0%

II. RESULTS

A. Minocycline Binding and Release from Hydroxysomes®

Minocycline attaches to Hydroxysomes™ at pH of 6.2-8.0. Minocyclineassociated with Hydroxysomes™ is unstable at lower than pH 5, forexample pH 4.7 or less. Therefore, minocycline releases fromHydroxysomes™ upon delivery into the skin.

B. Fluorescence Assessment of the Formulation

A Minocycline-HCl 0.1% (wt/wt) solution was prepared with 0.1 mL ofwater and 0.05 g of Hydroxysomes®. This was added to the formulationabove. The formulation was then applied on the filter paper to assessthe fluorescence emission. The fluorescence emission was assessedvisually with using a commercial black light (MR. LIGHT T5 14W, UV peakwave length around 365 nm) as an excitation source. The fluorescenceemission was not observed with the control sample without Hydroxysomes®.The minocycline-HCl sample in the cream without Hydroxysomes® emittedyellow-green fluorescence with the excitation of the black light. Theminocycline-HCl sample containing Hydroxysomes® emitted no fluorescence.

III. CONCLUSIONS

The above results demonstrate the development of the following:

-   1. Hydroxysomes® bind Minocycline HCl at neutral or alkaline pH and    release minocycline at acidic pH;-   2. Minocycline HCl-Hydroxysomes® substantially reduced the    fluorescence in the topical formulation.

VI. ADDITIONAL CHARACTERIZATION OF MINOCYCLINE-HYDROXYSOMES® COMPLEXESA. Minocycline Stability

Minocycline-Hydroxysomes® (25 mg/g) and minocycline alone (2.5 mg/mL)were added to water in glass vials and the vials capped and stored atroom temperature for 6 days. The vials were then centrifuged and thesupernatant analyzed by HPLC. The vial containing minocycline becamevisibly discolored during the test, and HPLC analysis revealedsubstantial degradation of minocycline with the occurrence of severalunidentified peaks were observed. In contrast, no discoloration wasobserved in the minocycline-Hydroxysomes® vial and no additional peaksother than minocycline were observed with HPLC analysis. See FIG. 1.

C. Minocycline Release at Skin pH

Minocycline-Hydroxysomes® (25 mg/g) were added to 0.5 M sodium acetatebuffer at pH 4.5. This buffer was used to represent the average pH ofstratum corneum. The suspension was mixed for 1 hour, centrifuged andthe supernatant analyzed for minocycline by HPLC. 92% of minocycline wasreleased under these conditions. The HPLC retention time of the releasedMNC was no different from the minocycline standard, indicating thatminocycline remained chemically unchanged during and after bound toHydroxysomes®. See FIG. 2

D. Minocycline Fluorescence

Minocycline-HCl and minocycline-Hydroxysomes® were imaged under whitelight and UV black light to detect fluorescence. Bulk powders ofminocycline and minocycline-Hydroxysomes® (25 mg MNC/g Hydroxysomes™)were placed under white and black light and photographs taken with adigital camera. Intense yellow-orange fluorescence was observed withminocycline powder, but no fluorescence was observed withMNC-Hydroxysomes®. See FIG. 3.

Minocycline and minocycline-Hydroxysomes® were then dispersed insilicone oil at 2% minocycline (w/v) and imaged by digital camera.Minocycline is not soluble in silicone oil so the fluorescence of thesolid material can be observed. Under white light, the yellow color ofminocycline was similar to that of minocycline-Hydroxysomes®. Underblack light, however, the strong yellow-orange fluorescence was stillobserved from minocycline, while low, diffuse fluorescence was observedfrom minocycline-Hydroxysomes®. The silicone oil control exhibited nofluorescence. Close-up images of the oil revealed intense fluorescencefrom the settled minocycline, in contrast to diffuse fluorescence on theminocycline-Hydroxysomes® surface. See FIG. 4.

E. Dermal Delivery of Minocycline in Human Skin

The dermal delivery of minocycline was determined at 2, 4 and 12 hours.For the 2-hour measurement, minocycline-Hydroxysomes® was applied to theskin surface in a static penetration cell. For the 4- and 12-hourmeasurements, minocycline-Hydroxysomes® was applied to the skin inflow-through diffusion (Franz) cells. Fresh human tissue was obtainedfrom surgery, maintained with growth supplements and antibiotics priorto the experiments and used within 7 days of receipt. To determine skinpenetration of minocycline, the skin surface was rinsed and the skinseparated into epidermis and dermis using the heat separation method.The epidermis and dermis were then extracted in 80% methanol and theextract analyzed for minocycline by HPLC.

The penetration of minocycline into skin from minocycline-Hydroxysomes®increased over time. By separating the skin into epidermis and dermis,the depth of penetration was determined by analyzing minocyclineconcentration in the separated tissue layers. Levels of minocycline werehigher in the epidermis and dermis at 12-hours compared to 4 hours.These results show that minocycline when delivered bound toHydroxysomes® penetrated into the deeper layers of the skin. Two (2) %of the applied minocycline was absorbed in skin after 12 hours. See FIG.5.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A topical minocycline composition comprising aporous, ceramic calcium phosphate particles having a pore size rangingfrom 2 nm to 100 nm and loaded with a minocycline active agent, whereinthe amount of minocycline active agent associated with the particlesranges from 0.1 to 100 mg/g and the composition is a formulationconfigured to be applied to a keratinized skin surface location of amammal and is chosen from a lotion, gel, cream and ointment, and furtherwherein the topical minocycline composition exhibits reducedfluorescence as compared to a control.
 2. The topical minocyclinecomposition according to claim 1, wherein the particles have a porevolume ranging from 30 to 85%.
 3. The topical minocycline compositionaccording to claim 1, wherein the particles are produced by: preparing afluid composition of calcium phosphate crystals; drying the fluidcomposition in a manner sufficient to produce precursor particles; andsubjecting the precursor particles to elevated temperatures in a mannersufficient to produce uniform, rigid, spherical nanoporous calciumphosphate particles.
 4. The topical minocycline composition according toclaim 1, wherein the formulation is a cream.
 5. The topical minocyclinecomposition according to claim 1, wherein the minocycline is bound tothe particles in an amount ranging from 1 to 50 mg/g.
 6. The topicalminocycline composition according to claim 1, wherein the particles havean average particle diameter of 2 microns or less.
 7. The topicalminocycline composition according to claim 1, wherein the compositioncomprises a minocycline salt.
 8. A method comprising applying to akeratinized skin surface location of a subject a topical minocyclinecomposition comprising porous, ceramic calcium phosphate particleshaving a pore size ranging from 2 nm to 100 nm and loaded with aminocycline active agent, wherein the amount of minocycline active agentassociated with the particles ranges from 0.1 to 100 mg/g and thecomposition is a formulation configured to be applied to a keratinizedskin surface location of a mammal and is chosen from a lotion, gel,cream and ointment, and further wherein the topical minocyclinecomposition exhibits reduced fluorescence as compared to a control. 9.The method according to claim 8, wherein the particles have a porevolume ranging from 30 to 85%.
 10. The method according to claim 8,wherein the amount of minocycline active agent associated with theparticles ranges from 1 to 50 mg/g.
 11. The method according to claim 8,wherein the particles have an average particle diameter of 2 microns orless.
 12. The method according to claim 8, wherein the subject is asubject that has acne and the method is a method of treating the subjectfor acne.
 13. The composition according to claim 1, wherein the calciumphosphate particles are chemically pure.
 14. The composition accordingto claim 1, wherein the particles are described by the molecular formulaCa₁₀(PO4)₆(OH)₂.
 15. The method according to claim 8, wherein thecalcium phosphate particles are chemically pure.
 16. The methodaccording to claim 8, wherein the particles are described by themolecular formula Ca₁₀(PO4)₆(OH)₂.
 17. The method according to claim 12,wherein the method further comprises assessing whether the subjectsuffers from acne.
 18. The composition according to claim 7, wherein theminocycline salt is minocycline hydrochloride.
 19. The compositionaccording to claim 1, wherein the composition further comprises a UVquencher/emission extinguisher.
 20. A method of treating acne in asubject, the method comprising: applying to a keratinized skin surfacelocation of a subject known to have acne a topical minocyclinecomposition comprising porous calcium phosphate particles having a poresize ranging from 2 nm to 100 nm and loaded with a minocycline activeagent, wherein the amount of minocycline active agent associated withthe particles ranges from 0.1 to 100 mg/g and the composition is aformulation configured to be applied to a keratinized skin surfacelocation of a mammal and is chosen from a lotion, gel, cream andointment, and further wherein the topical minocycline compositionexhibits reduced fluorescence as compared to a control.